The normal functioning of the adult nervous system relies critically on the proper structure and function of the vascular system. Blood vessels provide neurons with oxygen and nutrients and protect them from toxins and pathogens. Nerves, in turn, control blood vessel dilation and contraction and also heart rate. Key to this functional interdependence is an extraordinarily tight physical association between nervous and vascular systems. In the periphery, nerves and vessels often run parallel to one another and in the central nervous system neural activity and vascular dynamics are tightly coupled. Indeed, emerging evidence shows that some neurodegenerative diseases, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), once thought to be caused by intrinsic neuronal defects, are in fact initiated and perpetuated by vascular abnormalities. Despite these important connections between the nervous and vascular systems, little is known about how the nervous system becomes so closely aligned with the vascular system during development. In this proposal, we have established a simple system using developing mouse somatosensory peripheral target innervation as a model to study this question. In Aim1, we will dissect the molecular mechanisms underlying the organization of the tight nerve/vessel association, particularly focusing on the role of a recently identified ligand-receptor pair, Sema3E-Pelxin-D1. We will apply both in vitro assays and in vivo mouse genetics approaches to address the function of Sema3E-Pelxin-D1 signaling in establishing the nerve/vessel association. In Aim 2, we will identify and characterize the intracellular signaling mechanisms downstream of Sema3E-Pelxin-D1 in neurons and endothelial cells. Using a novel image-based RNAi genome-wide screen, we have identified and validated several potential candidates mediate Sema3E-Plexin-D1 signaling. We will characterize their roles in Sema3E-Plexin-D1 signaling and compare whether similar signaling mechanisms are used in Sema3E -mediated axon guidance and endothelial cell migration. Together, these proposed experiments will uncover cellular and molecular mechanisms underlying neuro-vascular interactions. These results may also improve our ability to diagnose, treat, and prevent neurological disorders that affect both neurons and vessels, including: peripheral neuropathies, Alzheimer's Disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS).